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Introduction: Bovine viral diarrhea virus (BVDV), a positive-sense single-stranded RNA virus, causes significant economic losses in the cattle industry. Current diagnostic methods for BVDV exhibit variable sensitivity and specificity, underscoring the need for more rapid and accurate detection approaches. Here, we developed a novel competitive ELISA (cELISA) to detect antibodies against the BVDV E2 protein., Methods and Results: We generated three monoclonal antibodies (mAbs)-3E6, 2D5, and 5B9-by immunizing mice with purified BVDV E2 protein expressed in Expi293F cells. Among these, mAb 3E6 displayed superior competitive binding abilities to the E2 protein, enabling effective differentiation between BVDV positive and negative sera. Remarkably, mAb 3E6 exhibited pan-genotypic recognition of various BVDV strains, including BVDV-1a, -1b, -1c, -1m, -1p, -1v, and -2a, while showing no cross-reactivity with the classical swine fever virus (CSFV). Computational modeling using AlphaFold 3 identified domain B of the E2 protein as the primary binding site for mAb 3E6. Building upon these findings, we established a cELISA employing mAb 3E6 and recombinant E2 protein. Receiver-operating characteristic (ROC) analysis revealed outstanding diagnostic performance, achieving a sensitivity of 99.26% and specificity of 98.99%. Further tests confirmed the cELISA's specificity for detecting BVDV-specific antibodies, with no cross-reactivity with antisera from animals infected or immunized against BCoV, BHV-1, BRV, AKAV, LSDV, BLV, and CSFV. Consistency was observed between results from the BVDV E2 cELISA and traditional virus neutralization test (VNT), demonstrating high sensitivity for monitoring antibody dynamics. In performance evaluations, the established cELISA exhibited high concordance with VNT in assessing 160 vaccinated sera and 190 clinical samples., Discussion: The BVDV E2 cELISA, utilizing mAb 3E6 to target domain B of the BVDV E2 protein, represents a reliable and effective serological diagnostic tool for the detection of antibodies against both BVDV-1 and BVDV-2. This methodology holds significant promise for applications in clinical diagnosis and the evaluation of vaccine efficacy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Qi, Wang, Le, Sun, Chang, Jiang, Yin and Pang.)

Numerous viruses, such as the bovine rotavirus (BRV), the bovine parvovirus (BPV), and the bovine viral diarrhea virus (BVDV), can cause bovine viral diarrhea syndrome. The global livestock industry has been subjected to significant consequences due to this condition. This results in considerable losses and hinders the production of crucial resources such as meat and milk, which are indispensable for sustaining the world's population. It is crucial to develop a quick and precise way of simultaneously detecting BVDV, BRV, and BPV, as they often occur together in mixed infections. A triplex loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) assay that can concurrently detect all three viruses was introduced in this study. The amplification process involved 30 minutes of incubation at 65 °C. The limits of detection (LODs) for BVDV, BRV, and BPV were 2.62 × 10 1 copies per μL, 2.43 × 10 1 copies per μL, and 2.50 × 10 1 copies per μL, respectively. The triplex LAMP-LFD assay was further evaluated in 156 anal swab samples, and the results were in agreement with the results of fluorescence quantitative PCR (qPCR) in more than 99% of the cattle. This assay is expected for the quick identification of triplex viruses in the field because it has high sensitivity and specificity and doesn't depend on laboratory equipment or conditions.

Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Since the start of the mandatory nationwide bovine viral diarrhea (BVD) eradication program in Germany in 2011, the number of persistently infected (PI) animals has decreased considerably, resulting in a continuous decrease in seroprevalence. The increasingly BVD-naive cattle population could facilitate spillover infections with non-BVDV ruminant pestiviruses. Here, we report two cases in which novel pestiviruses were isolated from cattle; in both cases, the whole genome sequence showed the highest level of identity to strain "Pestivirus reindeer-1". Both novel viruses gave positive results in BVDV diagnostic test systems, confirming that cross-reactivity is an important issue in pestivirus diagnostics. In the first case, the pestivirus was probably transmitted from sheep kept with the affected cattle, suggesting that the co-housing of small ruminants and cattle is a risk factor. The source of infection could not be determined in the second case. The occurrence of these two cases in independent cattle holdings within a relatively short time frame suggests that it would be useful to determine the presence of pestiviruses in small ruminants or even wild ruminants to better assess risk factors, especially for BVDV-free populations.

Bovine viral diarrhea virus (BVDV) is the cause of bovine viral diarrhea disease, one of the most economically important livestock diseases worldwide. The majority of BVD disease control programs rely on the detection and then elimination of persistent infection (PI) cattle, as the continuing source of disease. The main purpose of this study was to design and develop an accurate G-quadruplex-based aptasensor for rapid and simple detection of BVDV-1. In this work, we utilized in silico techniques to design a G-quadruplex aptamer specific for the detection of BVDV-1. Also, the rationally designed aptamer was validated experimentally and was used for developing a colorimetric biosensor based on an aptamer-gold nanoparticle system. Firstly, a pool of G-quadruplex forming ssDNA sequences was constructed. Then, based on the stability score in secondary and tertiary structures and molecular docking score, an aptamer (Apt31) was selected. In the experimental part, gold nanoparticles (AuNPs) with an average particle size of 31.7 nm were synthesized and electrostatically linked with the Apt31. The colorimetric test showed that salt-induced color change of AuNPs from red to purple-blue occurs only in the presence of BVDV-Apt31 complex, after 20 min. These results approved the specificity of Apt31 for BVDV. Furthermore, our biosensor could detect the virus at as low as 0.27 copies/ml, which is an acceptable value in comparison to the qPCR method. The specificity of the aptasensor was confirmed through cross-reactivity testing, while its selectivity was confirmed through plasma testing. The sample analysis showed 90% precision and 94% accuracy. It was concluded that the biosensor was adequately sensitive and specific for the detection of BVDV in plasma samples and could be used as a simple and rapid method on the farm., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rabiei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bovine Trypanosomiasis and other infectious diseases cause relevant loss for the livestock industry impacting productive/reproductive indices. This study intended to better understand the frequency, seasonality, and profile of infections associated with Bovine Trypanosomiasis. A total of 1443 serum samples were screened for T. vivax infection and other infectious diseases: Neosporosis, Leptospirosis, Bovine Leukosis Virus infection/(BLV), Infectious Bovine Rhinotracheitis/(IBR) or Bovine Viral Diarrhea/(BVD). Distinct methods were used for screening and diagnosis: immunofluorescence assay (Trypanosomiasis), ELISA (Neosporosis,BLV,IBR,BVD) and microscopic agglutination test (Leptospirosis). Our findings demonstrated that the seropositivity for Trypanosomiasis=57% was similar to Neosporosis=55%, higher than Leptospirosis=39% and BVL=34%, but lower than IBR=88% and BVD=71%. The seropositivity for Trypanosomiasis was higher in the autumn and lower in the winter. Regardless the season, the IBR seropositivity (min=73%;max=95%) was higher than Trypanosomiasis (min=48%;max=68%). Moreover, Neosporosis (min=71%;max=100%) and BVD (min=65%;max=76%) were more frequent than Trypanosomiasis in the summer, winter and spring. The diagnosis outcome revealed that Trypanosomiasis&IBR=43% and Trypanosomiasis&Neosporosis=35% were the most frequent co-infections with higher seropositivity in the autumn (58%) and summer (80%), respectively. Noteworthy, high seropositivity to Trypanosomiasis&BVD was registered in the autumn (46%). Together, our data re-enforce the relevance of differential diagnosis between Trypanosomiasis with other bovine infectious diseases and that differences in the seasonality profile is a relevant aspect to be considered while selecting the differential diagnosis to be applied., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bovine viral diarrhea virus (BVDV) is considered to be the most common agent of severe diarrhea in cattle worldwide, causing fever, diarrhea, ulcers, and abortion. Bovine herpesvirus 1 (BoHV-1) is also a major bovine respiratory disease agent that spreads worldwide and causes extensive damage to the livestock industry. Recombinase polymerase amplification (RPA) is a novel nucleic acid amplification method with the advantages of high efficiency, rapidity and sensitivity, which has been widely used in the diagnosis of infectious diseases. A dual RPA assay was developed for the simultaneous detection of BVDV and BoHV-1. The assay was completed at a constant temperature of 37 °C for 30 min. It was highly sensitive and had no cross-reactivity with other common bovine viruses. The detection rate of BVDV RPA in clinical samples (36.67%) was higher than that of PCR (33.33%), the detection rate of BoHV-1 RPA and PCR were equal. Therefore, the established dual RPA assay for BVDV and BoHV-1 could be a potential candidate for use as an immediate diagnostic., (© 2024. The Author(s).)

Since being reported in 1979 and 2006, indirect fluorescent antibody (IFA) tests have not been reported to detect bovine viral diarrhea virus (BVDV) antibodies to our knowledge. Thus, we re-evaluated the efficacy and usefulness of IFA tests for BVDV serology. We tested 4 combinations of 2 antibody conjugates (fluorescein isothiocyanate [FITC]-conjugated rabbit IgG anti-bovine IgG; rabbit IgG F(ab') 2 fragment anti-bovine IgG [F(ab') 2 FITC-IgG]) and 2 washing solutions (PBS; carbonate-bicarbonate-buffered saline [CBBS]) to evaluate the specificity of an IFA test for BVDV. We compared the sensitivity of the optimal combination with virus neutralization (VN) tests and an ELISA, and compared IFA with VN titers against different genotype (subgenotype) strains. For the F(ab') 2 FITC-IgG/CBBS combination, only 1 of the 156 (0.6%) 4-fold diluted cattle sera resulted in a nonspecific reaction; other combinations led to a much higher incidence (22.9-37.2%). For the F(ab') 2 FITC-IgG/CBBS combination, IFA detection rates were identical (36 of 59) for BVDV1 and BVDV2 genotypes, and IFA titers against them were strongly correlated ( r = 0.99). The antibody-detection rates of the IFA tests were almost identical to those of VN tests and the ELISA (κ: 0.96 and 0.89, respectively). The IFA titers against 4 strains (BVDV1a, BVDV1j, BVDV2a, and an unidentified strain) were similar, 1,024 to ≥4,096, although the VN titers were different. Thus, our IFA tests were specific and sensitive, and more useful than VN tests given that the IFA tests could evaluate the immune status of cattle using a representative strain, regardless of genotype (subgenotype)., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

With the rapid development of cattle industry, bovine viral diarrhea virus (BVDV) is becoming widespread in China, which causes serious economic losses to the industry. Effective vaccination and viral surveillance are critical for the prevent and control of BVDV infection. In the present study, the immunogenic domain of E2 protein of BVDV-1 was expressed by prokaryotic pET-28a vector. Monoclonal antibodies (mAbs) against E2 protein were prepared and systemically examined by western blot, immunofluorescence assay, blocking ELISA (bELISA) and virus neutralization test (VNT). The mAb 1E2B3, which showed good reactivity and neutralizing activity to BVDV-1 strains, was selected for ELISA establishment. After a series of screening and optimization, a novel bELISA for highly sensitive and specific detection of BVDV-1 antibodies was established, using HRP-labeled 1E2B3 and recombinant E2 protein. ROC analysis of 91 positive and 84 negative reference bovine serum samples yielded the area under the curve (AUC) of 0.9903. A diagnostic specificity of 96.43 % and a sensitivity of 95.6 % were achieved when the cutoff value was set at 24.31 %. There was no cross reaction to the positive sera of classical swine fever virus (CSFV), BVDV-2, border disease virus (BDV), bovine parainfluenza virus type 3 (BPIV3), infectious bovine rhinotracheitis virus (IBRV), foot-and-mouth disease virus (FMDV), Mycoplasma bovis (M.bovis) and Brucella. The total agreement rate of bELISA with VNT was 93.96 % (249/265). In addition, the result of bELISA was positively correlated with neutralizing antibody titer, and the bELISA could well distinguish the serum samples before and after BVDV vaccination. These results indicate that the established bELISA in this study is specific, sensitive, simple and convenient, which provides technical support for the vaccine efficacy evaluation, prevention and control of BVD in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

The review provides an analysis of literature data on the persistent form of Bovine Viral diarrhea/Mucosal disease (BVD) and is focused on virus and host factors, including those related to immune response, that contribute the persistence of the virus. BVD is a cattle disease widespread throughout the world that causes significant economic damage to dairy and beef cattle. The disease is characterized by a variety of clinical signs, including damage to the digestive and respiratory organs, abortions, stillbirths and other failures of reproductive functions.

The lethality of the bovine viral diarrhea virus (BVDV) in cattle involves inapparent infection and various, typically subclinical, syndromes. Cattle of all ages are vulnerable to infection with the virus. It also causes considerable economic losses, primarily due to reduced reproductive performance. In the absence of treatment that can completely cure infected animals, detection of BVDV relies on highly sensitive and selective diagnosis methods. In this study, an electrochemical detection system was developed as a useful and sensitive system for the detection of BVDV to suggest the direction of diagnostic technology through the development of conductive nanoparticle synthesis. As a countermeasure, a more sensitive and rapid BVDV detection system was developed using the synthesis of electroconductive nanomaterials black phosphorus (BP) and gold nanoparticle (AuNP). To increase the conductivity effect, AuNP was synthesized on the BP surface, and the stability of BP was improved by using dopamine self-polymerization. Moreover, its characterizations, electrical conductivity, selectivity, and sensitivity toward BVDV also have been investigated. The BP@AuNP-peptide-based BVDV electrochemical sensor exhibited a low detection limit of 0.59 copies mL -1 with high selectivity and long-term stability (retaining 95% of its initial performance over 30 days).

Bovine viral diarrhoea virus (BVDV) infection is a worldwide distributed animal disease. BVDV is the causal agent of congenital defects, diarrhea, reproductive failure, and contaminating biological products. Virus Neutralization test (VNT) as a gold standard method is used for detection of BVDV. Although this assay is very sensitive and specific, it has disadvantages including requires to an experienced person and cell culture facilities. VNT is time-consuming. It is important to design a method that does not have the mentioned disadvantages. So, in-house indirect enzyme linked immunosorbent assay (i-ELISA) was developed for laboratories where it is not possible to perform VNT. The system was made using NADL strain of BVDV and MDBK cell line. This ELISA system was compared with a commercial ELISA kit using 99 bovine sera. Coefficient of variation (CV) was calculated 3.9% and 4.8% for the positive and negative control, respectively for the designed i-ELISA system. The sensitivity, specificity, and accuracy of i-ELISA system was 88%, 53.6%, and 70.7% respectively. Based on our result correlation between in- house and commercial ELISA kit for detection of antibody against BVDV in bovine sera was significant (Kappa coefficient =0.41, p < 0.05). Results of the present study suggested that an in-house ELISA as an affordable and confident system for primary screening of the sera used for biological product., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Bovine viral diarrhoea virus (BVDV) causes substantial economic losses to the cattle industry; however, control and eradication can be achieved by identifying and removing persistently infected cattle from the herd. Each UK nation has separate control programmes. The English scheme, BVDFree, started in 2016 and is voluntary., Methods: We analysed the test results submitted to BVDFree from 5847 herds between 2016 and 2020., Results: In 2020, 13.5% of beef breeders and 20.0% of dairy herds that submitted tests had at least one positive (virus/antibody) test result. Although lower than in previous years, there was no clear trend in the proportion of positive tests over time. In virus testing herds, 0.4% of individual tests were positive in 2020, and 1.5% of individual tests were positive in BVDV-positive virus testing herds. Dairy herds and larger herds were more likely to join BVDFree, and dairy herds were also more likely to virus test than beef breeder herds. Larger herds, herds that used virus testing and herds that had BVDV-positive test results were more likely to continue submitting tests to BVDFree., Conclusions: The findings provide a benchmark for the status of BVDV control in England; continued analysis of test results will be important to assess progress towards eradication., (© 2022 The Authors. Veterinary Record published by John Wiley & Sons Ltd on behalf of British Veterinary Association.)

Background: Control programs were implemented in several countries against bovine viral diarrhea (BVD), one of the most significant cattle diseases worldwide. Most of the programs rely on serological diagnostics in any phase of the program. For the detection of antibodies against BVD virus (BVDV), neutralization tests as well as a variety of (commercially available) ELISAs are used. Here, test systems applied in various laboratories were evaluated in the context of an international interlaboratory proficiency trial. A panel of standardized samples comprising five sera and five milk samples was sent to veterinary diagnostic laboratories (n=51) and test kit manufacturers (n=3)., Results: The ring trial sample panel was investigated by nine commercially available antibody ELISAs as well as by neutralization tests against diverse BVDV-1, BVDV-2 and/or border disease virus (BDV) strains. The negative serum and milk sample as well as a serum collected after BVDV-2 infection were mostly correctly tested regardless of the applied test system. A serum sample obtained from an animal immunized with an inactivated BVDV-1 vaccine tested positive by neutralization tests or by total antibody or E rns -based ELISAs, while all applied NS3-based ELISAs gave negative results. A further serum, containing antibodies against the ovine BDV, reacted positive in all applied BVDV ELISAs, a differentiation between anti-BDV and anti-BVDV antibodies was only enabled by parallel application of neutralization tests against BVDV and BDV isolates. For the BVDV antibody-positive milk samples (n=4), which mimicked prevalences of 20% (n=2) or 50% (n=2), considerable differences in the number of positive results were observed, which mainly depended on the ELISA kit and the sample incubation protocols used. These 4 milk samples tested negative in 43.6%, 50.9%, 3.6% and 56.4%, respectively, of all investigations. Overall, negative results occurred more often, when a short sample incubation protocol instead of an over-night protocol was applied., Conclusions: While the seronegative samples were correctly evaluated in most cases, there were considerable differences in the number of correct evaluations for the seropositive samples, most notably when pooled milk samples were tested. Hence, thorough validation and careful selection of ELISA tests are necessary, especially when applied during surveillance programs in BVD-free regions., (© 2022. The Author(s).)

Background: Between 2007 and 2011 several thousands of calves died from bovine neonatal pancytopenia (BNP), a bleeding syndrome triggered by vaccine induced alloantibodies from the dams. Following withdrawal of the involved bovine viral diarrhoea virus (BVDv) vaccine, the incidence of this condition rapidly decreased, with no reported cases in the last 5 years. Here, we report a recent immune-mediated pancytopenia in three calves from two different suckler herds, clinically indistinguishable from BNP., Case Presentation: Three Belgian Blue suckler calves from two different farms, aged around two weeks, showed multiple bleedings disseminated on the skin and petechiae and ecchymoses on the mucosae. Blood examination confirmed anaemia, leukopenia and thrombocytopenia. BVDv infection was excluded. Despite blood transfusion and cortisone therapy, all three animals died. Necropsy and histology confirmed bone marrow depletion. Binding of IgG from the dams on leukocytes of the calves was demonstrated by flow cytometry. Two calves, originating from the same farm, received colostrum from the same dam. None of the calves were given colostrum replacers or colostrum supplements. No link with the BNP causing BVDv vaccine could be evidenced. However, dams had been vaccinated against bovine herpesvirus 1, parainfluenza-3 virus, bovine respiratory syncytial virus and bluetongue virus serotype 8., Conclusions: Alloimmune mediated pancytopenia was evidenced in three animals, clinically and pathologically indistinguishable from BNP. Whether this disease is again vaccine mediated remains to be determined., (© 2021. The Author(s).)

Bovine viral diarrhea virus (BVDV) infection results in a wide variety of clinical manifestations and is a pathogen that is able to cause huge economic losses in the cattle industry worldwide. It is important to identify cattle that are persistently infected (PI) by BVDV within the herd as early as possible because PI animals are the main reservoir of the virus. In contrast, cattle who are acutely infected (AI) with BVDV show various clinical signs, but most cattle show either mild symptoms or are asymptomatic. In general, AI and PI animals can be distinguished by repeat testing within an interval of at least 21 days. However, we found a rare case of a BVDV2-infected AI animal with long-term viral presence, making it indistinguishable from PI through two tests within an interval of 21 days. As a result, we diagnosed one infected animal as AI after 35 days from the initial sample collection via multiple analyses. Our findings recommend performing an additional test using samples that have been collected after 14-21 days from the second sample collection in cases where it is difficult to accurately differentiate an AI diagnosis from a PI diagnosis after only two tests. Additionally, our analysis exhibits that monitoring the number of copies of viruses with similar genomes in the sera by means of quantitative real-time RT-PCR through several sample collections periods might be useful to distinguish AI from PI. Furthermore, our data suggest that the AI animals with a long-term viral presence who show test results similar to those of PI animals might be the result of a coincidental combination of various factors that are present in cattle fields. These findings provide useful information that can be used to improve the diagnosis of BVDV in the field.

Pooled samples are used in veterinary and human medicine as a cost-effective approach to monitor disease prevalence. Nonetheless, there is limited information on the effect of pooling on test performance, and research is required to determine the appropriate number of samples which can be pooled. Therefore, this study aimed to evaluate the use of pooled serum samples as a herd-level surveillance tool for infectious production-limiting diseases: bovine viral diarrhoea (BVD), infectious bovine rhinotracheitis (IBR), enzootic bovine leukosis (EBL) and Neospora caninum (NC), by investigating the maximum number of samples one can pool to identify one positive animal, using commercial antibody-detection ELISAs. Four positive field standards (PFS), one for each disease, were prepared by pooling highly positive herd-level samples diagnosed using commercially available ELISA tests. These PFS were used to simulate 18 pooled samples ranging from undiluted PFS to a dilution representing 1 positive in 1,000 animals using phosphate-buffered saline as diluent. A 1:10 dilution of the PFS resulted in positive results for IBR, BVD and EBL. Moreover, for IBR and BVD, results were still positive at 1:100 and 1:30 dilutions, respectively. However, for NC, a lower dilution (8:10) was required for a seropositive result. This study indicates that, at herd-level, the use of pooled serum is a useful strategy for monitoring infectious diseases (BVD, IBR and EBL) but not NC, using readily available diagnostic assays., (© 2021. The Author(s).)

Aims: To explore recommendations that New Zealand veterinarians make for diagnosing and managing bovine viral diarrhoea (BVD) in cattle herds under different clinical scenarios and their opinions towards potential barriers and opportunities for implementing BVD control programmes in New Zealand., Methods: A cross-sectional survey of registered veterinarians in New Zealand was conducted in 2019. Respondents were asked about the approaches they would use to manage BVD under different clinical scenarios as well as their opinions on national BVD control. A subset of veterinarians completed a more in-depth survey providing additional free-text responses on a range of different BVD topics. Descriptive statistics were provided for all quantitative study variables and the free-text responses were also analysed to generate further insights into veterinarians' perceptions towards BVD management., Results: The cross-sectional survey was completed by 101 of an estimated 870 (11.6%) cattle veterinarians. Thirty-five veterinarians completed the in-depth survey. There was wide variation in the BVD diagnostic testing and vaccination protocols that respondents recommended under different clinical scenarios. Annual bulk milk BVD testing was perceived as a valuable tool for initiating BVD discussions with dairy farmers. Respondents indicated that beef farmers were more difficult to engage in BVD control largely due to the logistical challenges of yarding cattle at the appropriate times to implement interventions, with many farmers only contacting veterinarians after experiencing a BVD outbreak Most respondents (91/101; 90%) believed it was possible to eradicate BVD from New Zealand, but cited lack of farmer awareness and poor compliance with management recommendations as significant barriers. The measure with the most support for inclusion in a compulsory national eradication programme was requiring farmers to declare the status of their animals prior to sale while the least supported measure was requiring farmers to double fence boundaries to prevent nose-to-nose contact with neighbouring stock. Although respondents highlighted the need for farmers and industry to support any national eradication programme in order for it to be successful, there was also recognition that veterinarians could be more pro-active in engaging with farmers particularly in discussions around the economics of BVD., Conclusions and Clinical Relevance: While the survey respondents appeared to be highly supportive of BVD control, it was perceived that financial and logistical barriers existed that could impede farmer engagement. Further extension efforts may be needed to ensure that veterinarians are presenting clear and consistent recommendations about BVD management to farmers. Abbreviations : BVD: Bovine viral diarrhoea; NAIT: National Animal Identification and Tracing System; PI: Persistently infected.

Bovine viral diarrhea virus (BVDV) causes significant economic loss in cattle. Detection of persistently infected (PI) animals is an important control measure, but persistence of maternal antibodies may result in false-negative test results. We assessed the sensitivity and specificity of 2 antigen ELISAs (Idexx BVDV Ag/Serum Plus and BVDV PI X2) and a reverse-transcription real-time PCR (RT-rtPCR; Idexx RealPCR BVDV) assay for detecting PI calves. Ear notch samples were collected from 1,030 calves ~3, 10, 24, and 38 d old (days 3, 10, 24, and 38). All day 38 samples were tested using 2 antigen ELISAs and RT-rtPCR, and any calf that tested positive by any of these tests was blood sampled at ~100 d old (day 100) for antigen and antibody testing by ELISA; samples collected on days 3, 10, and 24 were tested using the antigen ELISAs and PCR. Calves were defined as PI if they were test-positive on day 38 by either ELISA or PCR and were antigen-positive on day 100. Twenty-six calves were PCR BVDV test-positive and one was BVDV PI X2 ELISA-positive at day 38. Five calves were defined as PI, and all tested positive by ELISAs and RT-PCR assay on days 3, 10, and 24. The sensitivity and specificity were 100% for both antigen ELISAs and 96.7% and 100%, respectively, by RT-rtPCR. Test results were not affected by calf age, suggesting that testing for PI calves can be undertaken at any age.

Bovine viral diarrhea virus (BVDV) is a viral pathogen associated with serious problems in the cattle industry. Cattle persistently infected (PI) with BVDV are mild or asymptomatic; however, they become a source of BVDV transmission to other cattle. Hence, it is important to rapidly identify and remove the PI animals from cattle herds. Whereas cattle acutely infected (AI) with BVDV have various symptoms, yet they generally recover within 3 weeks. However, there is a paucity of information concerning clinical characteristics of AI cattle. Further accumulation of information would be required to accurately diagnose AI cattle with BVDV. Here, we attempted to obtain valuable information via various analyses using a case report of BVD outbreak that occurred for approximately four months in Iwate Prefecture in 2017. Using eight calves and multiple tests (real-time RT-PCR, virus isolation, enzyme-linked immunosorbent assay, and virus neutralization assay) over 6 weeks, we diagnosed the continuous BVD outbreak as an acute infection and not a persistent one. Additionally, we revealed that the sporadic case was caused by low pathogenic BVDV2 via BVDV genotyping and phylogenetic analysis. The data suggest that BVDV2 AI animals might also be a source of transmission to susceptible calves; hence, it might persist for a long period owing to multiple AI animals. These findings provide useful information to diagnose AI and PI cattle with BVDV in the field.

In 2019, diarrhea cases occurred on cattle farms in Qionglai and Guang'an, Sichuan Province. Two out of 20 (10%) serum and nasal swab samples were positive when tested using a bovine viral diarrhea virus (BVDV) antigen-capture ELISA kit. Two non-cytopathic strains of BVDV were isolated and named QL1903 and GA190608, respectively. The nucleotide sequences of the genomes of the two isolates were 89.52% identical. Phylogenetic analysis based on the 5'-UTR sequence revealed that the BVDV isolate QL1903 belonged to BVDV subtype 1b, whereas isolate GA190608 clustered with strains HN1814, EN-19, and BJ09_26 in a separate branch, which has tentatively been classified as a new genetic subtype, "1v".

Bovine viral diarrhea (BVD) is a major worldwide disease with negative economic impact on cattle production. Successful control programs of BVD require the identification and culling of persistently infected (PI) animals with bovine viral diarrhea virus (BVDV). A variety of diagnostic tests are available to detect BVDV, but no comparison has been performed among those tests in Argentina. Sera collected from 2864 cattle, belonging to 55 herds from three Argentinean provinces, were analyzed by nested RT-PCR (RT-nPCR) to detect BVDV for diagnostic purposes. Additionally, this study evaluated the agreement of the RT-nPCR along with virus isolation, antigen-capture ELISA, and real-time RT-PCR for BVDV detection in archived bovine serum samples (n = 90). The RT-nPCR was useful for BVDV detection in pooled and individual serum samples. BVDV was detected in 1% (29/2864) of the cattle and in 20% (11/55) of the herds. The proportion of BVDV-positive sera was not statistically different among the tests. In addition, comparisons showed high agreement levels, with the highest values between both RT-PCR protocols. The frequency of BVDV infection at individual and herd level was lower than the reported values worldwide. Since follow-up testing was not performed, the frequency of PI cattle was unknown. Also, this study demonstrated that the four diagnostic tests can be used reliably for BVDV identification in individual serum samples. Further epidemiologically designed studies that address prevalence, risk factors, and economic impact of BVDV in Argentina will be necessary to implement effective control programs.

Detection of bovine viral diarrhea virus (BVDV) in aborted fetus samples is often difficult due to tissue autolysis and inappropriate sampling. Studies assessing different methods for BVDV identification in fetal specimens are scarce. The present study evaluated the agreement between different diagnostic techniques to detect BVDV infections in specimens from a large number of bovine aborted fetuses and neonatal deaths over a period of 22 years. Additionally, genetic, serological, and pathological analyses were conducted in order to characterize BVDV strains of fetal origin. Samples from 95 selected cases from 1997 to 2018 were analyzed by antigen-capture ELISA (AgELISA), nested RT-PCR (RT-nPCR), and real-time RT-PCR (RT-qPCR). In addition, amplification and sequencing of the 5'UTR region were performed for phylogenetic purposes. Virus neutralization tests against the BVDV-1a, BVDV-1b, and BVDV-2b subtypes were conducted on 60 fetal fluids of the selected cases. Furthermore, the frequency and severity of histopathological lesions were evaluated in BVDV-positive cases. This study demonstrated that RT-nPCR and RT-qPCR were more suitable than AgELISA for BVDV detection in fetal specimens. However, the agreement between the two RT-PCR methods was moderate. The BVDV-1b subtype was more frequently detected than the BVDV-1a and BVDV-2b subtypes. Neutralizing antibodies to any of the three subtypes evaluated were present in 94% of the fetal fluids. Microscopically, half of the BVDV-positive cases showed a mild non-suppurative inflammatory response. These results emphasize the need to consider different methods for a diagnostic approach of BVDV associated to reproductive losses.

Infection of bulls with bovine viral diarrhoea virus (BVDV) can result in the development of virus persistence, confined to the reproductive tract. These bulls develop a normal immune response with high neutralizing antibody titres. However, BVDV can be excreted in the semen for a prolonged period. Although relatively rare, in this study we describe six separate cases in bulls being prepared for admission to artificial breeding centres. Semen samples were tested in a pan-Pestivirus-reactive real-time PCR assay and viral RNA was detected in semen from five of the bulls for three to eight months after infection. In one bull, virus was detected at low levels for more than five years. This bull was found to have one small testis. When slaughtered, virus was only detected in the abnormal testis. The low levels of BVDV in the semen of these bulls were only intermittently detected by virus isolation in cell culture. This virus-contaminated semen presents a biosecurity risk and confirms the need to screen all batches of semen from bulls that have been previously infected with BVDV. The use of real-time PCR is recommended as the preferred laboratory assay for this purpose.

Introduction: Pestiviruses are the cause of reproductive problems, diseases of the gastrointestinal and respiratory tracts of animals. Three species are important for cattle: Pestivirus A, B, and H. Fast and reliable methods of differentiation of these pathogens are currently needed. Aims and objectives of the study: the development of multiplex real time PCR for the simultaneous detection and differentiation of three viruses., Material and Methods: The nucleotide sequences of the conserved regions of the 5´-UTR genes of pes tivirusesA, B, and H served as a target., Results: The reaction showed high specificity, sensitivity, reproducibility and was able to detect virus RNA at a concentration of not less than 0.6-1.2 lg TCID 50/cm 3 . Cross-reactions with other pestiviruses wer e not observed. Real time PCR confirmed the results obtained previously in RT-PCR with gel electrophoresis detection. In a parallel study of 1823 biological samples, the results of the two reactions were completely consistent. Pestivirus spp. was detectedin 76 samples, Pestivirus A was present in 73 samples, Pestivirus B - in 3 samples, and Pestivirus H was not detected., Discussion: A two-step real time PCR was developed for the simultaneous detection and differentiation of three pestiviruses. Modified pan primers of S. Vilcek et al. were used for the first reaction, and primers and probes of our own design were used for virus typing, which resulted in high reaction efficiency., Conclusion: On the big dairy farms for livestock maintenance, there are favorable conditions for the circulation of pathogenic viruses. In this situation, rapid diagnostic methods are needed to quickly identify of several viruses. Real-time triplex analysis can be recommended as the rapid method for mass epidemiological studies, as well as for screening fetal calf serum used for virus cultivation in medicine and veterinary practice., Competing Interests: The authors declare no conflict of interest.

In cattle, conceptus-derived interferon tau (IFNT) is the pregnancy recognition (PR) signal. Our previous studies showed that non-cytopathic bovine viral diarrhoea virus (ncpBVDV) infection inhibited IFNT-induced interferon stimulated gene (ISG) expression, potentially causing early embryonic death. This study investigated the effect of bovine viral diarrhoea virus (BVDV) infection on upstream regulatory pathways of ISG production using an established PR model. Uterine endometrial cells from 10 apparently healthy and BVDV free cows were cultured and treated with 0 or 100 ng/mL IFNT for 24 h in the presence or absence of ncpBVDV infection. Microarray and pathway analysis were used to determine the IFNT-induced upstream regulators. Expression of the genes associated with the identified pathways were quantified with qPCR. IFNT challenge activated the signalling pathways associated with IFN receptors, JAK1/TYK2, IRFs and STATs and ncpBVDV infection inhibited the activation of IFNT on this pathway. Inhibition of this upstream signalling pathway may thus reduce ISG production to disrupt maternal PR. In addition, the reduction of uterine immunity by ncpBVDV infection may predispose the animals to uterine infection, which in turn impairs their reproductive performance. This provides a mechanism of how BVDV infection leads to early pregnancy failure in cows.

Bovine viral diarrhea (BVD), one of the most important infectious diseases in cattle, causes major economic losses and significant impact on animal welfare worldwide. The major source for virus spread is persistently infected, immunotolerant calves and, therefore, their early identification is of utmost importance for disease prevention. Here, a ring trial was initiated to control the performance of diagnostic tests used in German regional laboratories in charge of the diagnostics within the country's BVD control program. A panel of five ear notch and five serum samples was provided for virological analysis. By an antigen ELISA, which was applied 26 times, the status of every sample was correctly identified in any case. In addition, a total of 54 real-time RT-PCR result sets was generated and also in most cases correctly classified. In addition to the virological test panel, a set of six sera and four milk samples was sent to the participating laboratories to be analyzed by serological methods. With serum neutralization tests, an excellent diagnostic sensitivity was achieved. However, one serum and both milk samples - positive for BVDV antibodies - repeatedly tested false negative by some of the used ELISA kits. All negative serum and milk samples were correctly identified by every commercial antibody ELISA. In conclusion, the BVDV proficiency test demonstrated that the used antigen/genome test systems allowed predominantly reliable diagnostics, while for four of the applied nine antibody ELISA kits adjustments are recommended., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The emergence of novel and divergent HoBi-like pestivirus (HoBiPeV) strains in cattle in Asia recently has raised concerns with regard to their reliable and accurate diagnosis. Hence, the aim of this study was to evaluate currently available BVDV diagnostic tests and HoBiPeV-specific diagnostic tests in detection of genetically divergent strains of HoBiPeV. One strain each of HoBiPeV-c and d were subjected to two BVDV diagnostic RT-PCR tests, one HoBiPeV specific RT-PCR test, three BVDV diagnostic qRT-PCR tests, one HoBiPeV specific qRT-PCR test and two BVDV antigen capture ELISAs. Archived cattle sera (n = 41) from farms with reports of HoBiPeV natural infection were assessed for detection of HoBiPeV antibodies by VNT and two commercial BVD antibody ELISA kits. BVDV diagnostic qRT-PCR tests had better sensitivity than BVDV diagnostic RT-PCR tests, while majority of them except a commercial kit showed a lower sensitivity for HoBiPeV-d strain. The HoBiPeV specific qRT-PCR test was found more sensitive than HoBiPeV specific RT-PCR but both had lower sensitivity for HoBiPeV-d strain, as displayed by primer/probe sequence mismatches. The BVDV E rns antigen ELISA detected both the strains of HoBiPeV, but with a lower sensitivity for HoBiPeV-d strain, whereas BVDV NS3 antigen ELISA failed to detect them even at a high HoBiPeV titre. Compared to VNT, commercial BVDV antibody ELISA showed low to moderate sensitivity in detection of HoBiPeV antibodies, with a failure rate of 31.25% for the whole virus antigen based ELISA and a failure rate of 56.25% for NS3 antibody ELISA. The present study demonstrated new challenges in HoBiPeV diagnosis indicating a need in improvement of both HoBiPeV specific diagnostic RT-PCR and qRT-PCR for better utility in HoBiPeV epidemiology and biological product safety. Although more studies are required, this study reinforces that combined use of BVDV E rns and NS3 antigen ELISA may have some utility in preliminary differentiation between HoBiPeV and BVDV infection in PI cattle. Additionally, we show that the comparative VNT has a better sensitivity in detection of HoBiPeV exposure and there is a need of robust antibody ELISA for reliable detection of antibodies against this emerging bovine pestivirus., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Eradicating bovine viral diarrhoea (BVD) from cattle populations requires a clear approach for determining the epidemiological status of individual herds and implementing the appropriate control measures to ensure the transmission cycle is cost-effectively broken. This is particularly important in countries such as New Zealand where there is currently no coordinated national programme and the herd-level decisions to control BVD are left to the discretion of individual farmers and veterinarians. To ensure greater consistency in the information being delivered by different stakeholders, we review the epidemiology of BVD in the context of New Zealand pastoral production systems and provides a series of simplified recommendations for the future control of BVD in beef and dairy herds. Based on analysis of BVD test accession data from commercial diagnostic laboratories, it has been estimated that 40.6% of dairy herds and 45.6% of beef herds tested had positive results for antibodies to BVD virus. While BVD continues to remain widespread and under voluntary control in New Zealand, it is recommended that herds test all individual mixed-age cows and replacement heifers for BVD virus or antigen and remove persistently infected animals from the breeding population. All new breeding animals that have entered the herd either through purchase or birth should also be tested for BVD virus. Biosecurity risks should be managed by reducing contacts with other herds and implementing targeted vaccination programmes. All individual purchased cattle should be tested and confirmed negative for BVD virus before being moved onto the buyer's property, even if the herd of origin had a negative antibody-based screening test. Herds should continue annual antigen or virus testing of all calves as soon as possible after birth to identify any persistently infected animals.

This is a cross-sectional study to assess the presence of antibodies in ruminants against selected pathogens associated with reproductive disorders in cattle in four Brazilian states, including the zoonotic agent Coxiella burnetii. The used tests were Virus Neutralization Assay for IBR and BVD, Microscopic Agglutination Test for Leptospira spp., Indirect Fluorescent Antibody Test (IFAT) for C. burnetii and Toxoplasma gondii, and Enzyme-Linked Immunosorbent Assay for Neospora caninum and Trypanosoma vivax. Seropositivity for C. burnetii was 13.7% with titers from 128 to 131,072; 57.8% for BoHV-1, with titers between 2 and 1,024; 47.1% for BVDV-1a, with titers from 10 to 5,120; 89.2% for N. caninum; 50% for T. vivax; and 52.0% for Leptospira spp., with titers between 100 to 800 (the following serovars were found: Tarassovi, Grippotyphosa, Canicola, Copenhageni, Wolffi, Hardjo, Pomona and Icterohaemorrhagiae); 19.6% for T. gondii with titer of 40. This is the first study that has identified C. burnetii in cattle associated with BoHV and BVDV, N. caninum, Leptospira spp., T. gondii and T. vivax. Thus, future studies should be conducted to investigate how widespread this pathogen is in Brazilian cattle herds.

The present study was aimed to establish a novel TaqMan real-time PCR (RTm-PCR) for detecting and typing bovine viral diarrhea virus (BVDV), and also to develop a diagnostic protocol which simplifies sample collection and processing. Universal primers and TaqMan-MGB probes were designed from the known sequences of conserved 5' - and 3'-untranslated regions (5'UTR, 3'UTR) of the NADL strain of BVDV. Prior to optimizing the assay, cDNAs were transcribed in vitro to make standard curves. The sensitivity, specificity and stability (reproducibility) were evaluated. The RTm-PCR was tested on the 312 feces specimens collected from persistently infected (PI) calves. The results showed the optimum conditions for RTm-PCR were 17.0 μmol/L primer, 7.5 μmol/L probe and 51.4°C annealing temperature. The established TaqMan RTm-PCR assay could specially detect BVDV without detecting any other viruses. Its detection limit was 1.55×100 copies/μL for viral RNA. It was 10000-fold higher than conventional PCR with excellent specificity and reproducibility. 312 samples were tested using this method and universal PCR from six dairy farms, respectively. Positive detections were found in 49 and 44 feces samples, respectively. The occurrence rate was 89.80%. In conclusion, the established TaqMan RTm-PCR could rapidly detect BVDV and effectively identify PI cattle. The detection limit of RTm-PCR was 1.55 copies/μL. It will be beneficial for enhancing diagnosis and therapy efficacy and reduce losses in cattle farms., (Copyright© by the Polish Academy of Sciences.)

The aim of this study was to investigate the persistent infection (PI) of bovine viral diarrhea virus (BVDV) along with its coexistence between BVDV antibody titer and BVD virus in blood of Holstein dairy cows. Only large commercial farms (each contained < 1000-3000 unvaccinated cows) were included. There were 11 dairy cattle herds. They included nearly 20,000 dairy cows. Totally, 140 cows, > 3 months to almost 10 years old, were randomly sampled. Indirect enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to detect BVDV antibody and virus, respectively. The percent positivity (PP) < 14 and ≥ 14 values are interpreted negative and positive, respectively. Simultaneously, whole blood samples pooled in groups of 10 animals were used for molecular detection of BVDV. The results revealed that 138 (98.56%) out of 140 cows were positive for BVDV antibody, while the BVDV antigen was detected only in 2 (1.42%) cows, which were negative for BVDV antibody and so were considered as persistent infection (PI) cows. They were also retested 3 weeks apart. Since the results showed the strong coexistence between seropositivity and BVD virus, in the infected dairy cattle herds, the combination of simple ELISA and pooled whole blood RT-PCR strategy could be an achievable approach to detect PI animals.

The outcomes of a voluntary bovine viral diarrhoea virus (BVDV) control programme that has been in place in the Netherlands since 1997 were analysed. This 'BVDV-free' programme was studied in dairy herds in the period 1 August 2007 to 1 August 2013. The programme was based on a test and cull approach at the herd level, after which the BVDV status was monitored by testing young stock for antibodies against BVDV or by antigen testing of newborn calves. One of the challenges of the programme was that, without any legislation or subsidies, farmers had to be motivated to pay all costs involved, with eradication of BVDV from their farm as the only incentive. During the study period, the percentage of dairy farms with a 'BVDV-free' status in the Netherlands increased from 13% to 24%, while the prevalence of active BVDV infections in Dutch dairy herds decreased. This may be related to the increasing number of participants in the 'BVDV-free' programme., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We investigated the potential for viremic sera from cattle persistently infected with bovine viral diarrhea virus to create false-negative antibody results when testing pools of 10 sera by indirect or blocking ELISAs. Seronegative viremic sera ( n = 23) were each added to a series of artificially constructed pools containing various percentages (0-90%) of antibody-positive sera, and the resulting pools were assayed for antibody. In all 23 cases, a negative antibody result was obtained in the pool containing no seropositive sera. In contrast, all pools containing ≥10% seropositive serum, representing a single seropositive animal in a pool of 10 samples, returned a positive result in both antibody ELISAs. We concluded that the likelihood of a false-negative antibody result occurring as a result of the presence of serum from a viremic animal was low, and therefore did not preclude the use of pooled sera for serosurveillance.

The pestiviruses bovine viral diarrhea virus 1 and 2 (BVDV-1 and -2, respectively) and HoBi-like pestivirus (HoBiPeV) are important pathogens of cattle, and a number of reverse-transcription PCR (RT-PCR)-based assays have been developed for their detection in clinical specimens. We evaluated a newly designed set of pan-bovine pestivirus primers (BP189-389) in a gel-based RT-PCR screening test for pestiviruses in the sera of beef calves destined for export from southern Brazil. Serum samples positive for BVDV antigens by an antigen ELISA ( n = 135) were submitted to RT-PCR assays using different sets of primers, followed by nucleotide sequencing of the amplicons. RT-PCR with pestivirus primers 324-326 detected 110 positive samples: BVDV-1 ( n = 62), BVDV-2 ( n = 38), and HoBiPeV ( n = 10). A PCR using primers HCV90-368 detected 97 positive samples (64 BVDV-1; 33 BVDV-2). An additional RT-PCR round using BVDV-2-specific primers (2F-2R) detected 45 positive samples (including 38 detected by primers 324-326 and 33 by HCV90-368); whereas a RT-PCR using HoBiPeV-specific primers (N2-R5) detected 26 positive samples (including 10 detected by primers 324-326).The assay using the primers BP189-389 detected all 135 ELISA-positive samples, including the 26 HoBiPeV detected by primers N2-R5. Our results demonstrated that primers BP189-389 compare favorably against other primer sets in the detection of bovine pestiviruses, especially HoBiPeV. This conventional PCR may be useful for efficient detection of pestiviruses in bovine sera and other specimens as well, especially in laboratories without real-time PCR equipment.

In late summer 2017, we observed acute, fatal cases of bovine viral diarrhea in captive Rocky Mountain bighorn sheep ( Ovis canadensis canadensis) in Colorado following use of a contaminated modified-live bluetongue virus vaccine. Following vaccination, at least 14 of 28 (50%) vaccinated bighorn sheep developed hemorrhagic diarrhea, and 6 of 28 (21%) vaccinated bighorn sheep died. Autopsy findings were predominantly necroulcerative-to-necrohemorrhagic gastrointestinal lesions. Less frequent lesions included suffusive hemorrhages of serosal surfaces of abdominal viscera, and lymphoid necrosis in gut-associated lymphoid tissues. All of the 6 bighorn sheep that died were positive on real-time PCR (rtPCR) for bovine viral diarrhea virus (BVDV) in multiple tissues. Seroconversion to BVDV-1 and immunohistochemistry for BVDV in affected tissues confirmed rtPCR results. Next-generation sequencing confirmed a match between the infecting strain of BVDV-1b and the contaminated vaccine.

Understanding risk factors for bovine viral diarrhoea (BVD) transmission is important for planning national disease control programmes. However, traditional statistical approaches may miss important features of BVD epidemiology due to the highly correlated nature of many farm-level risk factors. In this cross-sectional study, we used data collected from 304 cattle herds in New Zealand during 2015/2016 to compare the results from multivariable logistic regression with Bayesian network (BN) analysis. Blood samples from 15 heifers from each farm were pooled and analysed with an antibody ELISA test to classify BVD virus exposure status. Farmers were surveyed about their general management practices, knowledge about BVD, and risk factors for disease transmission, including onto- and off-farm movements, within- and between-farm contacts, and whether they implemented BVD control measures for their service bulls. Multiple imputation was used to infer missing values in the dataset prior to statistical analysis. The results showed that 57/116 (49.1%) beef farms and 95/188 (50.5%) dairy farms were likely to be actively infected with BVD virus. Almost 60% of farms had movements of heifers/cows onto the premises and 13.8% of farmers reported contact with cattle from other farms. The results of the multivariable logistic regression showed that farms where heifers/cows had been moved onto the premises during all or most of the past five years were at higher risk of being BVD seropositive than farms without those movements (OR 2.21, 95% CI 1.29-4.24). Farms where cattle had occasional or rare contacts with cattle on other farms were also at increased risk compared with farms without any animal contacts between farms (OR 2.63, 95% CI 1.33-5.41) although this association was not frequency-dependent. Only close animal contacts between farms was directly associated with BVD status in the BN model, however, this approach further untangled other complex associations between correlated management factors, and provided additional important insights into BVD epidemiology. Compared to other countries with intensive production systems, over the fence contact appeared to play a more important role in New Zealand pastoral-based production systems and should be considered when developing strategies for a national BVD control programme., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The major impacts of bovine viral diarrhoea (BVD) on cattle health and production have prompted many countries to embark on national elimination programmes. These programmes typically involve identifying and removing persistently infected (PI) cattle in infected herds and implementing biosecurity measures, such as pre- or post-movement testing. In order to design a systematic national control programme to eliminate BVD in New Zealand, which achieves the greatest benefits to the industries at the lowest cost to individual farmers, an accurate understanding is necessary of the epidemiology, economics and social motivation for BVD control in New Zealand. In this article we briefly review the pathogenesis of BVD, transmission and diagnosis of BVD virus infection, and effectiveness of vaccination. We summarise the current state of knowledge of the prevalence, risk factors for transmission, and financial impacts of BVD in New Zealand. We describe control programmes in Europe and then discuss the challenges that must be addressed to design a cost-effective national control programme to eliminate BVD in New Zealand.

Pestivirus bovine viral diarrhea virus type 1 species is responsible for cosmopolitan diseases affecting cattle and other ruminants, presenting a wide range of clinical manifestations, with relevant impact on zootechnic production. Understanding genomic characteristic and virus taxonomy is fundamental in order to sustain control and prophylactic programs. Given the recent various studies reporting a relatively high number of new strains, in particular from Asian countries, in the present study, four hundred-eighty-two genomic sequences have been evaluated applying the palindromic nucleotide substitutions method for genotyping. Based on the secondary structure alignment and computing genetic distance among strains in the 5' untranslated region of Pestivirus RNA, the current taxonomy of the species was reviewed. Twenty-two genotypes have been identified, applying a nomenclature based on divergence in the genus., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We performed a thorough fit-for-purpose evaluation of commercial ELISAs for the detection of bovine viral diarrhea virus (BVDV)-specific antibodies in serum and in milk by testing 2 panels of well-characterized serum and milk samples. Sixteen ELISAs from 9 different manufacturers, available on the Belgian market at the time of our study, were assessed for their diagnostic and analytical sensitivity (DSe and ASe, respectively), diagnostic specificity (DSp), and repeatability relative to the virus neutralization (VN) test considered to be the gold standard assay. Using serum as a matrix, DSe was much lower for competitive (c)ELISAs (min. 45%, max. 65%) than for indirect (i)ELISAs (min. 85%, max. 100%), partly because of the lower detection of positive samples from vaccinated animals included in the panel. ASe was also better for iELISAs; DSp was >95% for all but 2 ELISAs. Repeatability, expressed as coefficients of variation (CV) of optical densities, was generally good, although 3 ELISAs had a mean CV >10%. With milk samples, as observed for serum, DSe was lower for cELISAs (min. 57%, max. 75%) than for iELISAs (min. 61%, max. 89%), and DSp was high for all ELISAs (min. 94%, max. 100%). Both DSe and ASe were lower when testing milk samples compared to serum samples. These results confirm that serologic monitoring of BVDV-free herds should be performed using serum samples of unvaccinated animals to avoid interference of vaccination and to maximize the chance of detecting seroconversion linked to BVDV infection. Further investigations using a larger collection of field samples are recommended.

Since 2008, the Swiss veterinary service has been running a mandatory eradication program for Bovine Viral Diarrhea (BVD) that is focused on detecting and eliminating persistently infected (PI) animals. Detection was initially based on antigen testing from ear tag samples of the entire cattle population, followed by antigen testing of all newborn calves until 2012. Since then, bulk milk serology (dairy herds) and blood sample serology (beef herds) have been used for the surveillance of disease-free herds. From 2008 to 2012, the proportion of newborn PI calves decreased from 1.4% to less than 0.02%. However, this success was associated with substantial expenditures. The aim of this study was to conduct an economic evaluation of the BVD eradication program in the Swiss dairy sector. The situation before the start of the program (herd-level prevalence: 20%) served as a baseline scenario. Production models for three dairy farm types were used to estimate gross margins as well as net production losses and expenditures caused by BVD. The total economic benefit was estimated as the difference in disease costs between the baseline scenario and the implemented eradication program and was compared to the total eradication costs in a benefit-cost analysis. Data on the impact of BVD virus (BVDV) infection on animal health, fertility and production parameters were obtained empirically in a retrospective epidemiological case-control study in Swiss dairy herds and complemented by literature. Economic and additional production parameters were based on benchmarking data and published agricultural statistics. The eradication costs comprised the cumulative expenses for sampling and diagnostics. The economic model consisted of a stochastic simulation in @Risk for Excel with 20,000 iterations and was conducted for a time period of 14 years (2008-2021). The estimated annual financial losses in BVDV infected herds were CHF 85-89 per dairy cow and CHF 1337-2535 for an average farm, depending on the production type. The median net present value (NPV) was estimated at CHF 44.9 million (90% central range: CHF 13.4 million-69.4 million) and the break-even point to have been reached in 2015. Overall, the outcomes demonstrate that the Swiss BVD eradication program results in a net benefit for the dairy sector. These findings are relevant for planning similar BVD control programs in other countries., (Copyright © 2017. Published by Elsevier B.V.)

A single-step TaqMan® RT-qPCR was developed for the detection of bovine viral diarrhea virus type 1 (BVDV-1), an important pathogen of cattle worldwide. The assay was based on conserved 5'UTR sequences of Italian BVDV-1 isolates. In order to establish a diagnostic protocol which simplifies sample collection and processing, the assay was tested on a variety of biological specimens collected from persistently infected calves. The samples analyzed included PBMCs, plasma, dry blood, ear notch and hair bulb. Time and costs required for the analysis of each type of specimen were compared. The RT-qPCR, whose lower limit of detection was 100 copies of viral RNA (1 TCID50), correctly identified all PI animals, irrespective of the type of specimen. The highest copy numbers were obtained from the RNAs extracted from PBMCs, ear notches and hair bulbs. Hair bulb-supernatants directly used as a template allowed identification of all PI animals. In conclusion, based on time and cost evaluation, the most effective and efficient protocol was the one based on the direct analysis of hair bulb-supernatants, avoiding the RNA extraction step.

The objective of this study was to determine if precolostral blood samples are useful to detect apparent fetal infections with bovine viral diarrhea (BVD) and infectious bovine rhinotracheitis (IBR) viruses. A convenience sample of 317 sera from 50 Canadian herds was used in the study. Antibody level was measured using 2 commercial IBR and BVD ELISA kits. Precolostral status of sera was confirmed on 304 samples using serum gamma-glutamyl transferase activity. Postcolostral serum samples yielded a higher proportion of positive results to IBR (OR = 86; 95% CI: 17.8 to 415.7) and BVD (OR = 199.3; 95% CI: 41.7 to 952.3) than did precolostral samples. All positive precolostral serum samples ( n = 7 of 304) originated from calves born to vaccinated cows. Postcolostral positive serum samples ( n = 11 of 13) originated mostly (60%) from calves born to non-vaccinated cows. Precolostral serum sampling can detect apparent fetal infections in a herd.

Background: In an era of ubiquitous electronic collection of animal health data, multivariate surveillance systems (which concurrently monitor several data streams) should have a greater probability of detecting disease events than univariate systems. However, despite their limitations, univariate aberration detection algorithms are used in most active syndromic surveillance (SyS) systems because of their ease of application and interpretation. On the other hand, a stochastic modelling-based approach to multivariate surveillance offers more flexibility, allowing for the retention of historical outbreaks, for overdispersion and for non-stationarity. While such methods are not new, they are yet to be applied to animal health surveillance data. We applied an example of such stochastic model, Held and colleagues' two-component model, to two multivariate animal health datasets from Switzerland., Results: In our first application, multivariate time series of the number of laboratories test requests were derived from Swiss animal diagnostic laboratories. We compare the performance of the two-component model to parallel monitoring using an improved Farrington algorithm and found both methods yield a satisfactorily low false alarm rate. However, the calibration test of the two-component model on the one-step ahead predictions proved satisfactory, making such an approach suitable for outbreak prediction. In our second application, the two-component model was applied to the multivariate time series of the number of cattle abortions and the number of test requests for bovine viral diarrhea (a disease that often results in abortions). We found that there is a two days lagged effect from the number of abortions to the number of test requests. We further compared the joint modelling and univariate modelling of the number of laboratory test requests time series. The joint modelling approach showed evidence of superiority in terms of forecasting abilities., Conclusions: Stochastic modelling approaches offer the potential to address more realistic surveillance scenarios through, for example, the inclusion of times series specific parameters, or of covariates known to have an impact on syndrome counts. Nevertheless, many methodological challenges to multivariate surveillance of animal SyS data still remain. Deciding on the amount of corroboration among data streams that is required to escalate into an alert is not a trivial task given the sparse data on the events under consideration (e.g. disease outbreaks).

In 2011, an outbreak of severe vesicular disease occurred in the state of Pará, Amazon region. Besides proliferative or verrucous lesions, cattle showed atypical clinical signs such as diarrhea and leading to death. The animals were submitted to clinical, pathological and molecular diagnosis, and laboratory tests have confirmed the presence of Pseudocowpox virus (PCPV), a Parapoxvirus genus member, and have also found Bovine viral diarrhea virus-1 (BVDV-1), probably causing persistent infection. The results of molecular diagnostics, followed by sequencing data demonstrated the circulation of both viruses (PCPV and BVDV-1) in an area previously affected by another poxvirus, as Vaccinia virus.The cocirculation between PCPV and BVDV-1 indicates a major concern for animal health because the clinical presentation can be a severe disease. This is the first detection of PCPV in the Brazilian Amazon., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Infection due to bovine viral diarrhoea virus (BVDV) is endemic in most cattle-producing countries throughout the world. The key elements of a BVDV control programme are biosecurity, elimination of persistently infected animals and surveillance. Bovine viral diarrhoea (BVD) is a notifiable disease in Belgium and an official eradication programme started from January 2015, based on testing ear notches sampled during the official identification and registration of calves at birth. An antigen-capture ELISA test based on the detection of BVDV Erns protein is used. Ear notch sample may also be used to characterize the genotype of the calf when appropriate elution/dilution buffer is added. Both BVDV antigen-ELISA analysis and animal traceability could be performed., Methodology: With regards to the reference protocol used in the preparation of ear notch samples, alternative procedures were tested in terms of BVDV analytic sensitivity, diagnostic sensitivity and specificity, as well as quality and purity of animal DNA., Principal Findings/significance: The Allflex DNA Buffer D showed promising results in BVDV diagnosis and genome analyses, opening new perspectives for the livestock industry by the exploitation of the animal genome. Due to the high number of cattle involved in the Belgian official BVDV eradication programme based on ear notch tags sample, a large database on both BVDV status of newborn calves and cattle genome could be created for subsequent different uses (e.g. traceability, determination of parentage, genetic signatures throughout the genome associated with particular traits) evolving through a more integrated animal health., Competing Interests: Competing Interests: We have the following interests: Anke Kurz is employed by IFN Schönow GmbH. There are no patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Pestiviruses infect a wide variety of animals of the order Artiodactyla, with bovine viral diarrhea virus (BVDV) being an economically important pathogen of livestock globally. BVDV is maintained in the cattle population by infecting fetuses early in gestation and, thus, by generating persistently infected (PI) animals that efficiently transmit the virus throughout their lifetime. In 2008, Switzerland started a national control campaign with the aim to eradicate BVDV from all bovines in the country by searching for and eliminating every PI cattle. Different from previous eradication programs, all animals of the entire population were tested for virus within one year, followed by testing each newborn calf in the subsequent four years. Overall, 3,855,814 animals were tested from 2008 through 2011, 20,553 of which returned an initial BVDV-positive result. We were able to obtain samples from at least 36% of all initially positive tested animals. We sequenced the 5' untranslated region (UTR) of more than 7400 pestiviral strains and compiled the sequence data in a database together with an array of information on the PI animals, among others, the location of the farm in which they were born, their dams, and the locations where the animals had lived. To our knowledge, this is the largest database combining viral sequences with animal data of an endemic viral disease. Using unique identification tags, the different datasets within the database were connected to run diverse molecular epidemiological analyses. The large sets of animal and sequence data made it possible to run analyses in both directions, i.e., starting from a likely epidemiological link, or starting from related sequences. We present the results of three epidemiological investigations in detail and a compilation of 122 individual investigations that show the usefulness of such a database in a country-wide BVD eradication program., (Copyright © 2015 Elsevier B.V. All rights reserved.)